DataSheet1_An Integrative Multiomics Approach to Characterize Prebiotic Inulin Effects on Faecalibacterium prausnitzii.docx

Faecalibacterium prausnitzii, a major commensal bacterium in the human gut, is well known for its anti-inflammatory effects, which improve host intestinal health. Although several studies have reported that inulin, a well-known prebiotic, increases the abundance of F. prausnitzii in the intestine, the mechanism underlying this effect remains unclear. In this study, we applied liquid chromatography tandem mass spectrometry (LC-MS/MS)-based multiomics approaches to identify biological and enzymatic mechanisms of F. prausnitzii involved in the selective digestion of inulin. First, to determine the preference for dietary carbohydrates, we compared the growth of F. prausnitzii in several carbon sources and observed selective growth in inulin. In addition, an LC-MS/MS-based intracellular proteomic and metabolic profiling was performed to determine the quantitative changes in specific proteins and metabolites of F. prausnitzii when grown on inulin. Interestingly, proteomic analysis revealed that the putative proteins involved in inulin-type fructan utilization by F. prausnitzii, particularly β-fructosidase and amylosucrase were upregulated in the presence of inulin. To investigate the function of these proteins, we overexpressed bfrA and ams, genes encoding β-fructosidase and amylosucrase, respectively, in Escherichia coli, and observed their ability to degrade fructan. In addition, the enzyme activity assay demonstrated that intracellular fructan hydrolases degrade the inulin-type fructans taken up by fructan ATP-binding cassette transporters. Furthermore, we showed that the fructose uptake activity of F. prausnitzii was enhanced by the fructose phosphotransferase system transporter when inulin was used as a carbon source. Intracellular metabolomic analysis indicated that F. prausnitzii could use fructose, the product of inulin-type fructan degradation, as an energy source for inulin utilization. Taken together, this study provided molecular insights regarding the metabolism of F. prauznitzii for inulin, which stimulates the growth and activity of the beneficial bacterium in the intestine.

普拉梭菌（Faecalibacterium prausnitzii）是人体肠道内的主要共生细菌，以其显著的抗炎功效著称，可有效改善宿主肠道健康。尽管多项研究证实，经典益生元（prebiotic）菊粉（inulin）能够提升肠道内普拉梭菌的丰度，但该效应背后的具体分子机制仍有待阐明。本研究采用基于液相色谱串联质谱（LC-MS/MS）的多组学研究策略，对普拉梭菌选择性降解菊粉的生物学与酶学机制进行了系统探究。
首先，为明确该菌对膳食碳水化合物的底物偏好，我们对比了普拉梭菌在多种碳源中的生长表型，观察到其在菊粉培养基中呈现出选择性生长优势。随后，我们开展了基于LC-MS/MS的胞内蛋白质组与代谢组分析，以定量检测普拉梭菌以菊粉为唯一碳源培养时，其体内特定蛋白质与代谢物的表达/含量变化。值得注意的是，蛋白质组学分析结果显示，普拉梭菌中参与菊粉型果聚糖利用的推定蛋白——尤其是β-果糖苷酶与淀粉蔗糖酶——在菊粉诱导条件下显著上调表达。
为验证上述两类蛋白的功能，我们分别在大肠杆菌（Escherichia coli）中过表达了编码β-果糖苷酶与淀粉蔗糖酶的基因bfrA和ams，并证实了重组蛋白具备降解果聚糖的活性。此外，酶活实验进一步表明，胞内果聚糖水解酶可有效降解果聚糖ATP结合盒（ATP-binding cassette, ABC）转运蛋白摄取的菊粉型果聚糖。同时，我们发现当以菊粉为碳源时，普拉梭菌的果糖摄取活性可通过果糖磷酸转移酶系统转运体得到显著增强。胞内代谢组分析结果显示，普拉梭菌可将菊粉型果聚糖降解产生的果糖作为核心能源物质，支撑其菊粉代谢通路的运转。
综上，本研究系统揭示了普拉梭菌代谢利用菊粉的分子机制，为阐明菊粉如何促进肠道内这一有益共生菌的生长与活性提供了关键理论依据。